Michael Schwameis

Thrombin as a multi-functional enzyme. Focus on in vitro and in vivo effects.

Thrombin is the central protease in the coagulation cascade and one of the most extensively studied of all enzymes. In addition to its recognised role in the coagulation cascade and haemostasis, thrombin is known to have multiple pleiotropic effects, which mostly have been shown only in in vitro studies: it plays a role in inflammation and cellular proliferation and displays a mitogen activity on smooth muscle cells and endothelial cells, predominantly by activation of angiogenesis. In vivo , thrombin effects were examined in animal models of intravenous or intraarterial thrombin infusion. An extensive literature search regarding in vivo data showed that i) thrombin administered as a bolus causes microembolism, ii) thrombin infused slowly at steady-state conditions (up to 1.6 U/kg/min) leads to bleeds but not to intravascular clotting, iii) large quantity of thrombin infused at low rates (0.05 U/kg/min) does not have any measurable effect, and iv) thrombin increases vascular permeability leading to tissue damage. Although several decades of research on thrombin functions have provided a framework for understanding the biology of thrombin, animal and human studies with use of newer laboratory techniques are still needed to confirm the pleiotropic thrombin functions shown in in vitro studies.

Immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial

BACKGROUND Chikungunya is an emerging arthropod-borne disease that has spread from tropical endemic areas to more temperate climates of the USA and Europe. However, no specific treatment or preventive measure is yet available. We aimed to investigate the immunogenicity and safety of a live recombinant measles-virus-based chikungunya vaccine. METHODS We did a randomised, double-blind, placebo-controlled, active-comparator, phase 1, dose-escalation study at one centre in Vienna, Austria. Healthy men and women aged 18-45 years with no comorbidities were randomly assigned, by computer-generated block randomisation (block size of 14), to receive either one of three escalating doses of the measles-virus-based candidate vaccine (low dose [1·5 × 10(4) median tissue culture infection doses (TCID50) per 0·05 mL], medium dose [7·5 × 10(4) TCID50 per 0·25 mL], or high dose [3·0 × 10(5) TCID50 per 1·0 mL]), or the active comparator-Priorix. Participants were additionally block-randomised to receive a booster injection on either day 28 or day 90 after the first vaccination. Participants and study investigators were masked to group allocation. The primary endpoint was the presence of neutralising anti-chikungunya antibodies on day 28, as assessed by 50% plaque reduction neutralisation test. Analysis was by intention to treat and per protocol. This trial is registered with EudraCT, number 2013-001084-23. FINDINGS Between Nov 22, 2013, and Feb 25, 2014, we randomly assigned 42 participants to receive the low dose (n=12), the medium dose (n=12), or the high dose (n=12) of the measles-virus-based candidate vaccine, or Priorix (n=6), of whom 36 participants (86%; n=9, n=12, n=10, n=5, respectively) were included in the per-protocol population. The candidate vaccine raised neutralising antibodies in all dose cohorts after one immunisation, with seroconversion rates of 44% (n=4) in the low-dose group, 92% (n=11) in the medium-dose group, and 90% (n=10) in the high-dose group. The immunogenicity of the candidate vaccine was not affected by pre-existing anti-measles immunity. The second vaccination resulted in a 100% seroconversion for all participants in the candidate vaccine groups. The candidate vaccine had an overall good safety profile, and the rate of adverse events increased with vaccine dose and volume. No vaccination-related serious adverse events were recorded. INTERPRETATION The live recombinant measles-virus-based chikungunya vaccine had good immunogenicity, even in the presence of anti-vector immunity, was safe, and had a generally acceptable tolerability profile. This vaccine is the first promising measles-virus-based candidate vaccine for use in human beings. FUNDING Themis Bioscience GmBH.

VWF excess and ADAMTS13 deficiency: a unifying pathomechanism linking inflammation to thrombosis in DIC, malaria, and TTP

Absent or severely diminished activity of ADAMTS13 (A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motif, member 13) resulting in the intravascular persistence and accumulation of highly thrombogenic ultra large von Willebrand factor (UL-VWF) multimers is the pathophysiological mechanism underlying thrombotic thrombocytopenic purpura. Reduced VWF-cleaving protease levels, however, are not uniquely restricted to primary thrombotic microangiopathy (TMA), e. g. thrombotic thrombocytopenic purpura, but also occur in other life-threatening thrombocytopenic conditions: severely decreased ADAMTS13 activity is seen in severe sepsis, disseminated intravascular coagulation (DIC) and complicated malarial infection. The clinical relevance of these secondary thrombotic microangiopathies is increasingly recognised, but its therapeutic implications have not yet been determined. The presence of a secondary TMA in certain diseases may define patient groups which possibly could benefit from ADAMTS13 replacement or a VWF-targeting therapy. This short-review focuses on the role of UL-VWF multimers in secondary TMA and discusses the potential of investigational therapies as candidates for the treatment of TTP. In conclusion, prospective clinical trials on the effectiveness of protease replacementin vivo seem reasonable. Carefully selected patients with secondary TMA may benefit from therapies primarily intended for the use in patients with TTP.

Increased platelet aggregation and in vivo platelet activation after granulocyte colony-stimulating factor administration. A randomised controlled trial.

Granulocyte colony-stimulating factor (G-CSF) stimulates the bone marrow to produce granulocytes and stem cells and is widely used to accelerate neutrophil recovery after chemotherapy. Interestingly, specific G-CSF receptors have been demonstrated not only on myeloid cells, but also on platelets. Data on the effects of G-CSF on platelet function are limited and partly conflicting. The objective of this study was to determine the effect of G-CSF on platelet aggregation and in vivo platelet activation. Seventy-eight, healthy volunteers were enrolled into this randomised, placebo-controlled trial. Subjects received 5 μg/kg methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) or placebo subcutaneously for four days. We determined platelet aggregation with a whole blood impedance aggregometer with various, clinically relevant platelet agonists (adenosine diphosphate [ADP], collagen, arachidonic acid [AA], ristocetin and thrombin receptor activating peptide 6 [TRAP]). Filgrastim injection significantly enhanced ADP (+40%), collagen (+60%) and AA (+75%)-induced platelet aggregation (all p<0.01 as compared to placebo and p<0.001 as compared to baseline). In addition, G-CSF enhanced ristocetin-induced platelet aggregation (+18%) whereas TRAP-induced platelet aggregation decreased slightly (-14%) in response to filgrastim. While baseline aggregation with all agonists was only slightly but insignificantly higher in women than in men, this sex difference was enhanced by G-CSF treatment, and became most pronounced for ADP after five days (p<0.001). Enhanced platelet aggregation translated into a 75% increase in platelet activation as measured by circulating soluble P-selectin. G-CSF enhances platelet aggregation and activation in humans. This may put patients suffering from cardiovascular disease and cancer at risk for thrombotic events.

A randomized, placebo-controlled phase I study assessing the safety and immunogenicity of a Pseudomonas aeruginosa hybrid outer membrane protein OprF/I vaccine (IC43) in healthy volunteers.

Introduction: IC43 is a recombinant outer membrane protein-based vaccine against Pseudomonas aeruginosa (P. aeruginosa) consisting of OprF- and OprI- epitopes (Opr, outer membrane protein; OprF/I, OprF/OprI hybrid vaccine) with an N-terminal His 6 tag (Met-Ala-(His)6-OprF190–342-OprI21–83). Objectives: The study aimed to confirm the optimal dose of IC43 in adults with regard to immunogenicity, safety, and tolerability after vaccination with three different dosages of IC43, compared with placebo, and to investigate a potential immune-enhancing effect of the adjuvant, aluminum hydroxide. Subjects were randomly allocated in a 1:1:1:1:1 ratio to one of five treatment groups: 50, 100, or 200 µg IC43 with adjuvant, 100 µg IC43 without adjuvant, or placebo (0.9% sodium chloride) and two intramuscular injections were given in the deltoid region 7 d apart. Results: The primary immunogenicity analysis of OprF/I-specific IgG antibody titers on day 14 demonstrated statistically significant differences among treatment groups (P < 0.0001), with a significantly higher immune response detected in each IC43 treatment group compared with placebo. From day 0 to day 14, a ≥4-fold increase in OprF/I-specific immunoglobulin G (IgG) antibody titers were observed in >90% of subjects in all IC43 treatment groups in the per-protocol (PP) and intention-to-treat (ITT) populations; a ≥ 50-fold titer increase was observed in 42.6% subjects including all IC43 treatment groups. On day 90, OprF/I-specific IgGs started to decline in all IC43 treatment groups but remained significantly higher until 6 mo compared with placebo. Assessment of functional antibody induction by opsonophagocytic assay (OPA) followed a similar pattern compared with OprF/I-specific IgG kinetics. At day 14, a ≥2-fold increase in OPA titer was observed in 54.5% subjects within all IC43 treatment groups. An increase in antibody avidity index was observed after the second vaccination. At day 14, >96% of subjects in each IC43 treatment group had detectable OprF/I-specific IgG antibodies. Anti-histidine IgG antibody titers peaked on day 14 and were reduced on day 90 in all IC43 treatment groups. OprF/I-specific IgG secreted by antibody-secreting cell (ASC) was detected in all IC43 groups by B-cell ELIspot after the second vaccination and up to 6 mo. All vaccinations were safe and well tolerated up to the maximum cumulative dosage of 400 µg IC43. Conclusion: IC43 doses equal to or greater than 50 µg were sufficient to induce a plateau of IgG antibody responses in healthy volunteers. Higher doses, whether adjuvanted or non-adjuvanted, were not more effective. Methods: In this phase I, randomized, placebo-controlled, observer-blinded, multicenter clinical trial, 163 healthy volunteers (18−65 y) were randomly assigned to five treatment groups (1:1:1:1:1). Three groups received IC43 with adjuvant: 50 µg (n = 32), 100 µg (n = 33), or 200 µg (n = 33). One group received IC43 100 µg without adjuvant (n = 32), and one group received placebo (0.9% sodium chloride) (n = 33). Each subject received two intramuscular vaccinations, separated by a 7-d interval (days 0 and 7) (Fig. 1). Humoral immune response was assessed by measurement of outer membrane protein F/I (OprF/I)-specific antibodies determined by enzyme-linked immunosorbent assay (ELISA), anti-histidine antibodies determined by ELISA, and functional antibody activity determined by opsonophagocytic assay (OPA), up to 6 mo post-vaccination. Antibody avidity was measured on days 7 and 14 from samples that had detectable vaccine antibody-specific immunoglobulin G (IgG) antibody titers. At the Austrian site only, the B-cell ELIspot assay was used to determine specific ASC responses. Safety was assessed using adverse event monitoring and clinical laboratory tests. Local and systemic tolerability was recorded in a subject diary for 7 d after each vaccination and by investigators up to 6 mo post-vaccination. Clinical trial registration number: NCT00778388

Morphine decreases ticagrelor concentrations but not its antiplatelet effects: a randomized trial in healthy volunteers

Our recent drug interaction trial with clopidogrel shows that morphine decreases the concentrations and pharmacodynamic effects of clopidogrel, which could lead to treatment failure in susceptible individuals.

Chikungunya vaccines in development

ABSTRACT Chikungunya virus has become a global health threat, spreading to the industrial world of Europe and the Americas; no treatment or prophylactic vaccine is available. Since the late 1960s much effort has been put into the development of a vaccine, and several heterogeneous strategies have already been explored. Only two candidates have recently qualified to enter clinical phase II trials, a chikungunya virus-like particle-based vaccine and a recombinant live attenuated measles virus-vectored vaccine. This review focuses on the current status of vaccine development against chikungunya virus in humans and discusses the diversity of immunization strategies, results of recent human trials and promising vaccine candidates.

Cardiac arrest due to accidental hypothermia—A 20 year review of a rare condition in an urban area

BACKGROUND Accidental hypothermic cardiac arrest is associated with unfortunate prognosis and large studies are rare. We therefore have performed an outcome analysis in patients that were admitted to Vienna University Hospital with the diagnosis of accidental hypothermic cardiac arrest. METHODS This study employed a retrospective outcome analysis of prospectively collected data in a selected cohort of hypothermic cardiac arrest patients. We screened 3800 cardiac arrest patients, treated at our department between 1991 and 2010, for eligibility. Inclusion criteria were cardiac arrest with a body core temperature ≤28 °C and return of spontaneous circulation. RESULTS A total of 18 patients who achieved return of spontaneous circulation were analysed. Nine patients (50%) achieved survival in good neurologic condition (defined as cerebral performance category CPC 1 or 2). Accidental hypothermia with consecutive cardiac arrest was caused by intoxication in most cases (67%). These patients had a better outcome than patients with other causes of accidental hypothermic cardiac arrest (OR=28; 95%KI 2-37.9; p<0.01). Hypothermia associated typical ECG changes after return of spontaneous circulation (Osborne waves) were more frequent in the surviving population (OR 16; 95%KI 1.3-19.5; p=0.05). CONCLUSIONS Accidental hypothermic cardiac arrest in a central European urban area is rare. Prognosis was excellent in patients where hypothermic cardiac arrest was caused by intoxication.

Effects of prasugrel on platelet inhibition during systemic endotoxaemia: a randomized controlled trial

P2Y(12) receptor antagonists have become a mainstay for the treatment of CVD (cardiovascular diseases). However, they have rarely been evaluated under pathophysiological conditions apart from arterial diseases. We hypothesized interactions between prasugrel and enhanced vWF (von Willebrand Factor) release in a model of systemic inflammation, and compared the pharmacodynamic effects of prasugrel against placebo on agonist-induced platelet aggregation and shear-induced platelet plug formation. A total of 20 healthy male volunteers were enrolled in a double-blind placebo-controlled two-way crossover trial. Each volunteer received either placebo or a 60 mg loading dose of prasugrel 2 h before endotoxin or placebo infusion. Platelet inhibition was measured with MEA (multiple electrode aggregometry), the PFA-100 system and the VASP (vasodilator-stimulated phosphoprotein) phosphorylation assay. Prasugrel blunted various platelet aggregation pathways, including those induced by ADP (-81%), AA (arachidonic acid) (-60%), ristocetin (-75%; P<0.001 for all) and, to a lesser degree, collagen or TRAP (thrombin-receptor-activating peptide). Prasugrel decreased shear-induced platelet plug formation, but vWF release during endotoxaemia partly antagonized the inhibitory effect of prasugrel as measured with the PFA-100 system. Endotoxaemia acutely decreased ristocetin and TRAP-induced platelet aggregation, and enhanced ristocetin-induced aggregation after 24 h. Strong in vivo blockade of P2Y(12) inhibits a broad spectrum of platelet aggregation pathways. However, vWF release may reduce prasugrels effects under high-shear conditions.

Reversal strategy in antagonizing the P2Y12 -inhibitor ticagrelor.

Patients on antiplatelet therapy have a higher incidence of bleeding complications. Reversal of antiplatelet drug effects is an important issue at trauma or emergency departments. For old and conventional anticoagulants, reversal strategies are established. While the effects of ticagrelor are reversible, developing a method to restore platelet function in patients is of importance due to its longer half‐life (approximately 8 h), compared with other P2Y12‐inhibitors.